Cytotoxic T-lymphocyte antigen as cysteine protease inhibitor

ABSTRACT

Disclosed are molecules which inhibit the proteolytic activity of cysteine proteases such as Cathepsin H, Cathepsin L and papain, and methods for using molecules which have the biological properties of cytotoxic T-lymphocyte antigen for inhibiting cysteine proteases and inhibiting proteoglycan degradation.

This application is a continuation in part application of U.S.application Ser. No. 07/915,923, filed on Jul. 17, 1992, now abandoned,and a 371 of PCT/US93/06552, filed Jul. 15, 1993.

FIELD OF THE INVENTION

The present invention relates to methods for inhibiting the bio-activityof enzymes. More specifically, the invention comprises molecules havingcytotoxic T-lymphocyte antigen properties, and methods of inhibiting theproteolytic activity of cysteine protease using such molecules.

DESCRIPTION OF THE RELATED ART

Cytotoxic T-Lymphocyte Antigen-2 (abbreviated CTLA-2) is a moleculeexpressed by activated T-cells and mast cells. Complementary DNA (cDNA)for two distinct but homologous forms of CTLA-2 are known, namelyCTLA-2α and CTLA-2β (Denizot et al., 1989, Eur. J. Immunol.,19:631-635).

The present specification describes the identification of the geneencoding for peptides with CTLA-2 activity, and the expression,purification and characterization of such peptides having CTLA-2activity. The identified gene for CTLA-2β codes for a protein consistingof 138 amino acids (15,900 g/mole) including a putative leader sequence.Removal of the hydrophobic N-terminus results in a protein of 110 aminoacids (12,800 g/mole). There are a total of five cysteine residues inthe molecule, two of which are in the putative leader sequence.Therefore, the mature protein should contain three cysteine residuesindicating the formation of disulfide linked dimers.

The CTLA-2α gene codes for a protein which is 98% homologous (90%identical) to CTLA-2β at the protein level. CTLA-2α has a total of threecysteines in the full-length protein but only one in the mature form.Therefore, CTLA-2α also has the potential of forming a disulfide linkeddimer.

The presently disclosed invention includes methods for inhibitingcertain cysteine proteases with molecules having CTLA-2α and CTLA-2βactivity. Members of the cysteine protease family all have cysteine intheir active sites, and the family includes the enzyme papain found inthe papaya plant, a developmentally regulated proteinase in theDictyostelium slime mold, Chinese goosberry actinidin, and the mammalianlysosomal Cathepsins B, H, and L (Portnoy et al., 1986, J. Biol. Chem.,261:14697).

Cathepsins are proteolytic enzymes found in most mammalian cells andtheir functions include cellular autolysis and tissue degredation(Bohley et al., 1992, Experimentia, 48:151; Funabiki et al., 1990, Int.J. Biochem. 22:1303). Cathepsin L is a major lysosomal protease and isresponsible for bulk turnover of intracellular protein. The cDNAsequence for CTLA-2β is known to be 40% homologous to the pro-region ofmouse Cathepsin L (Denizot et al., id.), but there is no homology to theactive protease sequence.

The present disclosure for the first time provides experimental evidenceindicating that a protein homologous to the pro-region of a cysteineprotease acts as an inhibitor of that cysteine protease. Specifically,the results hereinbelow show, inter alia, that purified CTLA-2β inhibitsthe proteolytic activity of the cysteine proteases papain, Cathepsin Hand Cathepsin L.

There is a need in the art for inhibitors of cysteine proteases as theseenzymes have been implicated in the formation of new foci of metastaticcarcinoma (Aoyama et al., 1990, Biochem. 87:8296; Keren et al., 1988,Cancer Res., 48:1416; Sloane et al., 1984, Cancer Metastases Rev. 3:249;Stearns et al., 1990, Arch. Biochem. Biophys., 283:447). Inhibition ofcysteine proteases is reported to be a good candidate for cancer therapy(Nakajima et al., 1991, Cancer Biology, 2:115).

In addition, there is further data implicating the role of cysteineproteases in the pathology of a number of diseases including: rheumatoidarthritis (Trabandt et al., 1990, Matrix, 10:349); glomerulonephritis(Baricos et al., 1991, Arch. Biochem. Biophys., 288:468); emphysema(Manson et al., 1986, Biochem. J., 233:925); osteoporosis (Delaisse etal., 1991, Biochem. J. 279:167); and Alzheimer's disease (Cole et al.,1989, Neurochemical Res., 14:933). Cysteine protease inhibitors shouldprove useful in the treatment of such disease states.

There have been a number of different synthetic organic compoundsproposed as inhibitors of cysteine proteases and cathepsins, such asdipeptidyl aldehydes (Sasaki et al., 1990, J. Enzyme inhib., 3:13);C-terminal diazomethyl ketones (Wikstrom et al., 1989, Arch. Biochem.Biopys., 270:286); andtrans-epoxysuccinyl-L-leucylamido-(4-guanido)butane, referred to as"E-64" (Baricos et al., Biochem. Biophys. Res. Comm., 155:1318).

However, such compounds have been found to be toxic as well asteratogenic (Fukushima et al., 1990, Toxicology and AppliedPharmacology, 185:1; Chen et al., 1989, Acta Paediatr. Jpn., 31:685;Tachikura, 1990, Acta Paeditr. Jpn. 32:495; Doherty et al., 1989, Exper.Cell Res., 185:506; Ivy et al., 1990, Lipofuscin and Ceroid Pigments,Plenum press, New York, pg. 31; Daston et al., 1991, Teratology,43:253).

CTLA-2α and CTLA-2β are naturally occuring mammalian proteins and arenot known to be either toxic or teratogenic Furthermore,physiologically, the CTLA-2 proteins act extracellularly in theinhibition of secreted cathepsins, and should not inhibit: cathepsinsfound intracellularlywithin lysosomes. Therefore, the normal functionsof the cathespins inside cells should not be affected.

SUMMARY OF THE INVENTION

The present invention provides for the use of peptides as inhibitors ofproteases and in particular specifically embody the peptides listedbelow.

The present invention provides for a polypeptide of the amino acidsequence:

Tyr-Ser-Leu-Asp-Glu-Glu-Arg-His-Arg-Arg-Leu-Met-Trp-Glu-Glu-Asn-Lys-Lys-Lys-Ile-Glu-Ala-His.(p117, SEQ ID NO. 5)

The present invention provides for a polypeptide of the amino acidsequence:

Ser-Leu-Asp-Asn-Glu-Trp-Lys-GLu-Trp-Lys-Thr-Thr-Phe-Ala-Lys-Ala-Tyr-Ser-Leu-Asp-Glu-Glu.(p118, SEQ ID NO. 6)

The present invention provides for a polypeptide of the amino acidsequence:

Glu-Asn-Lys-Lys-Lys-Ile-Glu-Ala-His-Asn-Ala-Asp-Tyr-Glu-Arg-Gly-Lys-Thr-Ser-Phe-(CYS).(p089, SEQ ID NO. 7)

The present invention provides for a polypeptide of the amino acidsequence:

Cys-Arg-Gly-Glu-Met-Ala-Pro-Asp-Leu-Pro-Glu-Tyr-Glu-Asp-Leu-Gly. (p092,SEQ ID NO. 8)

The present invention provides for a polypeptide of the amino acidsequence:

R R A V W E K N M K M I E L H N. (SEQ ID NO. 9)

The present invention provides for a polypeptide of the amino acidsequence:

R R A I W E K N M R M I Q L H N. (SEQ ID NO. 10)

The present invention provides for a polypeptide of the amino acidsequence:

R R L M W E E N K K K I E A H N. (SEQ ID NO. 11)

The present invention provides for a polypeptide of the amino acidsequence:

Thr-Leu-Thr-Phe-Asp-His-Ser-Leu-Glu-Ala-Gln-Trp-Thr-Lys-Trp-Lys-Ala-Met-His-Asn-Arg-Leu-Tyr-Gly-Met-Asn-Glu-Glu-Gly-Trp-Arg-Arg-Ala-Val-Trp-Glu-Lys-Asn-Met-Lys-Met-Ile-Glu-Leu-His-Asn-Gln-Glu-Tyr-Arg-Glu-Gly-Lys-His-Ser-Phe-Thr-Met-Ala-Met-Asn-Ala-Phe-Gly-Asp-Met-Thr-Ser-Glu-Glu-Phe-Arg-Gln-Val-Met-Asn-Gly-Phe-Gln-Asn-Arg-Lys-Pro-Arg-Lys-Gly-Lys-Val-Phe-Gln-Glu-Pro-Leu-Phe-TyrGlu (SEQ ID NO. 12).

The present invention also provides for methods of inhibitingproteolytic activity of cysteine protease with the above listedpeptides. In a preferred embodiment said cysteine protease is selectedfrom the group consisting of Cathepsin H, Cathepsin L, Cathepsin B andpapain. In a preferred embodiment the inhibiting peptide retains theactivity of CTLA-2 and is substantially identical to the peptidedescribed in SEQ ID NO. 4. In another preferred embodiment theinhibiting peptide is substantially identical to that described as p117(SEQ ID NO. 5).

The present invention also provides for a method for inhibiting thedegradation of proteoglycans with polypeptides. In a prefered embodimentthe inhibiting polypeptide is selected from the group consisting of thepolypeptides listed above. In a preferred embodiment the inhibitingpeptide is substantially identical to that described as p117 (SEQ ID NO.5).

The present invention also provides a method for inhibiting thedegradation of proteoglycans in cartilage organ cultures byadministering of an effective amount of an inhibiting peptide werein theinhibiting peptide is selected from the group listed above. In apreferred embodiment the inhibiting peptide is substantially identicalto that described as p117 (SEQ ID NO. 5).

The present invention thus provides for compounds and methods useful forthe treatment of diseases associated with degradation of proteoglycans,or the activity of cysteine proteases. Being protein based, thecompounds of the present invention are advantageous in that there wouldbe less toxicity when compared with the activity of chemical drugs thatmay inhibit some cysteine proteases. The present compounds are thususeful in themselves and as lead compounds for the development ofderivatives with improved biological activity.

The invention comprises molecules with CTLA-2 activity, includingbioactivites associated with CLTA-2α and CTLA-2β. Such molecules includeisolated, purified recombinant proteins, synthetic analogs, oligopeptidefragments of the full lenath CTLA-2 proteins, and quarternary structuralanalogs such as dimeric or tetrameric forms of the CTLA-2 protein. In apreferred embodiment the protein is in a monomeric or dimeric form.

The invention also includes methods of inhibiting the enzyme activity ofcysteine proteases using the above-stated molecules, such methods beingadaptable by those skilled in the art for use as therapeutics fortreatment of disease states whose causative agent is abnormal orexcessive cysteine protease activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elution profile for crude baculovirus supernatantchromatographed on Superdex-75. Protein concentration is plotted vs.fraction number. The position of protein standards is shown.

FIGS. 2(A) and 2(B) show purification of CTLA-2β by Superdex 75 gelfiltration chromatography monitored by SDS-PAGE analysis of Superdex-75purification fractions. FIG. 2(A) is a coomassie brilliant blue R-250stain and FIG. 2(B) is a transfer to ProBlot for Western analysis.

FIGS. 3(A) and 3(B) show SDS-PAGE analysis of Q-sepharose anion-exchangepurification fractions of CTLA-2β as monitored by SDS-Page. (10%-20%tricine under non-reducing conditions.) Gel was stained for totalprotein with coomassie brilliant blue R-250 as shown in 3(A) ortransferred to ProBlot for western analysis as shown in 3(B).

FIGS. 4(A)-FIG. 4(D) depict purification of CTLA-2β on reverse-phasechromatography, with: 4(A) showing elution profiles for monomeric and4(B) showing dimeric species on a Vydac C4 reverse phase colum; and,showing the analysis for the monomer and dimer fractions monitored bySDS-PAGE, FIG. 4(C) shows coomassie stain and FIG. 4(D) shows a westernblot. Dimer fractions 26-34 are on the Left and monomer fractions 26-34are on the Right for each gel.

FIGS. 5(A)-5(C) depict Circular Dichoism (CD) spectra of purifiedCTLA-2β. Each spectra is plotted as mean residual ellipticity vs.wavelength from 260 to 195 nm, with: FIG. 5(A) showing CTLA-2β monomerafter Q-Sepharose chromatography, FIG. 5(B) showing CTLA-2β monomerafter C4 reverse phase chromatography, FIG. 5(C) showing CTLA-2β dimerafter C4 reverse phase chromatography.

FIGS. 6(A)-6(D) graphically depict inhibition of Cathepsin L by CTLA-2β,the hydrolysis of Z-Phe-Arg-AMC by cathepsin L was measured byfluoresence at the concentration of monomer or dimer indicated: A plotof the fluoresence due to Cathepsin L activity vs. time for differentconcentrations of CTLA-2β, with data for both the monomer, FIG. 6(A),and the dimer, FIG. 6(B), shown, and also the crude fluorescence data asdepicted in (A) and (B) analyzed on a Dixon plot of the form v-/v+ vs.CTLA-2β concentration are shown in 6(C) and 6(D).

FIG. 7 shows the determination of the Ki value for the interaction ofCTLA-2β with Cathepsin L using a Dixon plot of the form 1/V vs. CTLA-2βconcentration. The concentration of Z-Phe-Arg-AMC used was 5 uM (closedcircles) and 2.5 uM (open circles). Linear regression was performed todetermine the Ki.

FIGS. 8(A) and 8(B) show the determination of the I.C. 50 values for theinhibition of papain and Cathepsin H using the Dixon plot of the formV-/V+ vs. CTLA-2β concentration, with: 8(A) showing data for theinhibition of Cathepsin H by CTLA-2β, using H-Arg-AMC as substrate, and8(B) showing data for the inhibition of Papain by CTLA-2β usingZ-Phe-Arg-AMC as the substrate.

FIGS. 9(A) and 9(B) graphically depict inhibition of Cathepsin L usingthe synthetic p117 peptide [SEQ ID No. 5], the rate of AMC released wasmeasured from linear regression of plots on nmoles AMC released overtime. Dixon plots of the form V-/V+ vs. peptide concentration were madeand the i.c. 50 determined, with: 9(A) showing the data for theinhibition of Cathepsin L by p117, and 9(B) showing a Dixon plot derivedfrom the data of 9(A). The various symbols plot different concentrationsof peptide tested (in uM).

FIGS. 10(A)and 10(B) graphically depict inhibition of Cathepsin L usingthe p092 peptide [SEQ ID No. 8] as in FIG. 9, with: 10(A) showing thedata for the inhibition of Cathepsin L by p092, and 10(B) showing aDixon plot derived from the data of 10(A). The various symbols plotdifferent concentrations of peptide tested (in uM) as labelled.

FIGS. 11(A) and 11(B) graphically depict inhibition of Cathepsin L usingthe p089 peptide, [SEQ ID No. 7] as in FIG. 9, with: 11(A) showing thedata for the inhibition of Cathepsin L by p092, (open box 0.0 uM, closedbox 1 uM, open circle 10 uM, closed circle 25 uM peptide) and: 11(B)showing a Dixon plot derived from the data of 11(A). (open box 0.0 uM,closed box 10 uM, and open circle 35 uM peptide). The rate of AMCreleased was measured from linear regression of plots on nmoles AMCreleased over time.

FIG. 12 is a graph showing inhibition by p117 peptide, of proteoglycanrelease from Cartilage Culture as tested by MOCA (Micro Organ CartilageCulture Assay). The results are shown as % inhibition of proteoglycanrelease.

DETAILED DESCRIPTION OF THE INVENTION

The following describes the production of recombinant (murine) CTLA-2βin insect cells using a baculovirus overexpression system (Summers etal., 1987, A Manual of Methods for Baculovirus Vectors and Insect CellCulture Procedures, Texas Ag. Exp. Sta. Bull. No. 1555); thepurification of the overexpressed CTLA-2β using column chromatographicsteps; and the demonstration that this recombinant protein, andfragments thereof, are inhibitors of the cysteine proteases such ashuman kidney cell cathepsins (Cathepsin H and Cathepsin L) and papainfrom the papaya plant.

EXAMPLE 1 Expression

The CTLA-2β gene was isolated from an eukaryotic expression cDNA librarymade from mRNA expressed by a murine T-ceIl hybridoma designated "AS-9"(Takashi Maki, New England Deaconess Hospital, Boston, Mass.).

This cDNA library was used in the expression screening protocoldescribed in Aruffo et al., 1987, Proc. Natl. Acad. Sci. (USA), 84:3365,and Aruffo et al., 1987, EMBO J., 6:3313. This expression screeningprotocol yielded a molecular clone as shown below in SEQ ID NO. 1.

In order to express the recombinant protein in the baculovirus system,the putative leader sequence was removed. The SER residue, indicated bythe underline, was changed to a MET residue to direct the synthesis ofthe recombinant protein in the baculovirus system. The protein sequenceof the CTLA-2β predicted from the cloned gene is (SEQ ID NO. 1):

                            Leu Asp Asn Lys Val Leu Val Ser Ile Cys Glu Gln                               Lys Leu Gln His                                                               1               5                   10                                              15                                                                      Phe Ser Ala Val Phe Leu Leu Ile Leu Cys Leu Gly                               Met Met Ser Ala                                                                           20                  25                                              30                                                                          Ala Pro Ser Pro Asp Pro Ser Leu Asp Asn Glu Trp                               Lys Glu Trp Lys                                                                       35                  40                                                45                                                                            Thr Thr Phe Ala Lys Ala Tyr Ser Leu Asp Glu Glu                               Arg His Arg Arg                                                                   50                  55                  60                                Leu Met Trp Glu Glu Asn Lys Lys Lys Ile Glu Ala                               His Asn Ala Asp                                                               65                  70                  75                                              80                                                                  Tyr Glu Arg Gly Lys Thr Ser Phe Tyr Met Gly Leu                               Asn Gln Phe Ser                                                                               85                  90                                              95                                                                      Asp Leu Thr Pro Glu Glu Phe Arg Thr Asn Cys Cys                               Gly Ser Ser Met                                                                           100                 105                                             110                                                                         Cys Arg Gly Glu Met Ala Pro Asp Leu Pro Glu Tyr                               Glu Asp Leu Gly                                                                       115                 120                                               125                                                                           Lys Asn Ser Tyr Leu Thr Pro Gly Arg Ala Gln Pro                               Glu                                                                               130                 135                 140   

The following synthetic oligonucleotide fragments were used as primersto generate a subfragment of CTLA-2β via polymerase chain reactionamplification:

5'Primer: (SEQ ID NO. 2)

GGGGGATCCA TGGCTGCTCC ATCC

3'Primer: (SEQ ID NO. 3)

TGTGGACCTT CCCGAGTCGG TCTCATTCGA TCGCCTAGGG GG

The generated subfragment was digested with NcoI and BamHI, and thetruncated fragment was subsequently ligated into a PBL 1392 vector(Invitrogen, San Diego, Calif.) for high-level expression in thebaculovirus/insect cell system (Invitrogen, CA.).

This construct contained the coding sequences for the mature form ofCTLA-2β protein (i.e. without the putative leader sequence and with anadditional methionine (MET) residue added at the proposed start of themature protein, and had the following amino acid sequence: (SEQ ID NO.4)

    Met Ala Ala Pro Ser Pro Asp Pro Ser Leu Asp Asn Glu Trp Lys Glu               1               5                   10                  15                    Trp Lys Thr Thr Phe Ala Lys Ala Tyr Ser Leu Asp Glu Glu Arg His                           20                  25                  30                        Arg Arg Leu Met Trp Glu Glu Asn Lys Lys Lys Ile Glu Ala His Asn                       35                  40                  45                            Ala Asp Tyr Glu Arg Gly Lys Thr Ser Phe Tyr Met Gly Leu Asn Gln                   50                  55                  60                                Phe Ser Asp Leu Thr Pro Glu Glu Phe Arg Thr Asn Cys Cys Glu Ser               65                  70                  75                  80                Ser Met Cys Arg Gly Glu Met Ala Pro Asp Leu Pro Glu Tyr Glu Asp                               85                  90                  95                    Leu Gly Lys Asn Ser Tyr Leu Thr Pro Gly Arg Ala Gln Pro Glu                               100                 105                 110                   

An alternate method for constructing the clone would be to use theabove-mentioned oligonucleotide primers to generate the same CTLA-2βfragment directly from cDNA made from a suitable murine T-cell line suchas KB5C20.

In order to optimize the growth of the SF9 cells and the expression ofthe recombinant CTLA-2β, a number of different multiplicity ofinfections (hereinafter "M.O.I.") and harvest times were tried. AnM.O.I. of 1 and 10 were used, and for both M.O.I.'s a sample wasanalyzed for CTLA-2β expression at 48, 72, and 96 hours after infection.According to Western analysis of the supernatants and cell pellets, thebest expression of secreted CTLA-2β was obtained at a M.O.I. of 10 witha 96 hour harvest time.

EXAMPLE 2 Purification of CTLA-2β

(i) Crude Supernatant: Baculovirus cell culture supernatant wassupplemented with 1 mM PMSF, 5 mM each of benzamidine and EDTA, 5 μg/mleach of chymostatin, antipain, E-64, and aprotinin, 2.μg/ml leupeptin,and 1 μg/ml pepstatin. The solution was adjusted to pH 7.5 and clarifiedby centrifugation for 30 min. at 10K rpm. Material was stored at -20° C.until needed.

(ii) Superdex-75 Gel-Filtration Chromatography: All further purificationoperations were performed in a cold room at 2° C. A Pharmaciasuperdex-75HR 16/60 FPLC column was equilibrated in 50 mM Tris-HCI (pH8.0) and 150 mM NaCl (Buffer A) and run at a flow rate of 1.5 ml/min.The Pharmacia FPLC system was set up to inject and run automatically. Avolume of 4 ml of crude supernatant was applied and the column elutedisocratically in a total volume of 135 ml of equilibration buffer.Fractions (2.25 ml) were collected and analyzed by duplicate SDS-PAGE;which were either stained for total protein or analyzed for CTLA-2βthrough a Western blot (see Protein Analysis below) . The fractions thatcontained CTLA-2β were pooled.

(iii) Q-Sepharose Anion-Exchange Chromatography: A Pharmacia 10/10C-column containing 10 ml of Q-Sepharose was equilibrated in Buffer B(50 mM Tris-HCI pH 8.0, 25 mM NaCl) at a flow rate of 1.0 ml/min. TheCTLA-2β pool from step (ii) was dialyzed against Buffer B using6,000-8,000 molecular weight cut-off membrane (Spectrum), and loadedonto the Q-sepharose column. The column was then washed with threecolumn volumes of Buffer B, and CTLA-2β was eluted subsequently in a 40ml linear gradient of 0.5M NaCl in Buffer B at a flow rate of 0.75ml/min. Fractions (1 ml) were collected and analyzed as in (ii).Fractions containing CTLA-2β were pooled and stored at 40C.

(iv) Reverse-phase Chromatography: reverse-phase chromatography wasperformed on a Beckman System Gold HPLC using a C4 column (Vydac, 25×1.0cm). 2.5 ml (1.0 mg) of CTLA-2β from step (iii) was injected onto the C4column previously equilibrated in 100 mM sodium acetate pH 6.5 at a flowrate of 2 ml/min. Material was eluted with a linear gradient of 5% to80% acetonitrile over 45 min. Fractions (2 ml) were collected andanalyzed as above.

EXAMPLE 3 Protein Characterization

(i) Protein Determination: Protein concentrations were determined eitherby the Pierce dye binding assay using BSA as a standard, or by theabsorbance at 280 nm using an extinction coefficient of 2.7×10⁴ L/mol/cm(calculated by using the amino acid composition data to quantify theamount of protein)

(ii) SDS-PAGE: Protein samples were analyzed by SDS-PAGE on 10-20%tricine buffered acrylamide gels (Novex) under non-reducing conditions.Protein was detected with Coomassie Brilliant Blue R-250. Western blotswere developed with affinity purified anti-peptide antibodies which weremade to either the middle of the protein, (amino acids 69-88 of themature sequence), or the C-terminal region (113-128).Alkaline-Phosphatase conjugated goat anti-Rabbit antibody was used asthe secondary antibody, and the blot was developed according to standardprotocols.

(iii) Secondary Structure: The secondary structure of CTLA-2β wasdetermined using circular dichroism (CD). CTLA-2β from steps (iii) and(iv) of Example 2 (in PBS buffer) were analyzed using an AVIV associatescircular dichroism spectrophotometer model 62DS. The spectra werecorrected for the absorbance of the buffer.

(iv) Molecular Weight: The molecular weights of the monomeric ordiimeric forms of CTLA-2β were determined by mass spectrometry. CTLA-2βfrom step (iv) of Example 2 was analyzed using a Finigan LASERMATLaser-Desorbtion Time-of-Flight Mass Spectrophotometer.

EXAMPLE 4 Cysteine Protease Inhibition

(i) Cathepsin L Assay: The capacity of CTLA-2β to inhibit the activityof the proteolytic enzyme Cathepsin L was measured. A fluorescence basedassay was set up according to Barrett and Kirschke (Barrett et al.,1981, Meth. in Enz., 80:535).

The enzyme sample was diluted with 750 μl of assay buffer (340 mM sodiumacetate--60 mM acetic acid--4 mM disodium EDTA, pH 5.5 and 8 mM DTTadded fresh) and various concentrations of CTLA-2β (a finalconcentration of 5-50 nM), was added. After an incubation period of 6minutes at room temperature, 1500 μl of 0.1% Brij 35 was added. Thesubstrate Z-Phe-Arg-AMC (AMC is aminomethyl-coumarin) was added at afinal concentration of either 2.5 μM or 5.0 μM.

The fluorescence of the free aminomethyl-coumarin was measured in an SLM4800s fluorimeter by excitation at 370 nm and emission at 432 nm over asix minute time period. Fluorescence was plotted vs. concentration ofCTLA-2β at the two substrate concentrations specified. In order tocalculate the velocities, a linear least squares analysis was performedover the initial linear part of the data. The fluorescence of thereaction mixture was measured both with and without CTLA-2β present inorder to determine the velocity with (V+) and without (V-) inhibitor.

The i.c. 50 values were determined from Dixon plots of the form V-/V+vs. I. In order to calculate the Ki value for the interaction of CTLA-2βwith Cathepsin L, 1/V was plotted vs. the concentration of CTLA-2β. TheKi value is equal to the negative of the X-axis intercept that gives thesame velocity at the different substrate concentrations if there iscompetitive inhibition (Dixon et al., 1979, Enzymes, 3rd Ed., AcademicPress, New York, page, 365).

(ii) Cathepsin H Assay: Cathepsin H was diluted in 750 μl of assaybuffer (200 mM KH₂ PO₄ --200 mM Na₂ HPO₄ --4 mM disodium EDTA pH 6.8 and40 mM Cysteine added fresh). CTLA-2β was added at a final concentrationrange of 5-200 nM, and reaction incubated as for Cathepsin L. Themixture was diluted with Brij 35 as in above, and the substrateH-Arg-AMC added at a final concentration of 5 μM. The fluorescence wasmeasured as above, and the i.c. 50 determined.

(iii) Papain assay: The assay conditions used were identical to that ofCathepsin L with the exceptions that the final concentration of enzymeused was 33.3 ng/ml, the final concentration of CTLA-2β was 5-100 nM,and that the assay buffer was 88 nM KH₂ PO₄ --12 MM Na₂ HPO₄ --1.33 mMdisodium EDTA 2.7 mM Cysteine added fresh. The fluorescence was measuredand the i.c. 50 was determined.

RESULTS

A) Baculovirus Expression.

After performing the time course and multiplicity of infectionexperiments, the expression level of CTLA-2β was approximately 50 mg/Lof conditioned media. Most of the expressed CTLA-2β was found in thecell culture supernatant and not inside the cells (data not shown). Thisobservation is interesting because the construct used for the expressiondid not have the putative leader sequence attached and has an additionalmethionine residue at the N-terminus to direct the synthesis. Themajority of the expressed CTLA-2β was monomeric, but some dimer form wasobserved.

B) Protein Purification.

Purification of the monomeric and dimeric forms of CTLA-2β wasaccomplished by a two-step protocol and yielded 2.5 mg of monomericCTLA-2β per 168 ml culture (See Table 1, below).

                  TABLE I                                                         ______________________________________                                        PURIFICATION OF CTLA-2β MONOMER                                                    VOLUME            TOTAL       TOTAL                                 STEP      (ml)     OD280/ml OD 280                                                                              mg/ml mg                                    ______________________________________                                        CELL CULTURE                                                                  SUPERNATANT                                                                             168.0    12.84    2157.1                                                                              2.97.sup.a                                                                          498.5                                 SUPERDEX 75                                                                             410.0    0.057    23.37 0.023.sup.a                                                                         9.43                                  Q-SEPHAROSE                                                                             6.0      0.87     5.23  0.424.sup.b                                                                         2.54                                  ______________________________________                                         Purification of CTLA2β from a 168.0 ml Baculovirus cellculture           supernatant.                                                                  .sup.a Concentration determined by Pierce assay.                              .sup.b Concentration determined using an extinction coefficient of 2.67       × 10.sup.4 L/mol/cm.                                               

The original cell-culture supernatant was applied to a Superdex-75Gel-Filtration column with most contaminants being removed in this step(see FIGS. 1 and 2). Several species that differed in their molecularweight were identified by western analysis (FIG. 2). Fractions 26-34 hada 75Kd molecular weight species which was visible only undernon-reducing conditions. This 75Kd species disappeared if the sample wasanalyzed on a reducing SDS-PAGE. The exact nature of this species isunknown but it may represent cross-reactivity of the antibodies.Fractions 40-46 contained a band on the western blot of 27,000 g/mole;corresponding to a dimeric form of CTLA-2β. However, according to theelution profile of protein standards, the molecular weight of a proteineluting in this position should be approximately 45,000 g/mole,corresponding to a tetramer of CTLA-2β. Therefore, the covalently linkeddimeric form of CTLA-2β probably forms a tetramer under non-denaturingconditions. Fractions 48-52 contained a band on the western blot of13,000 g/mole, corresponding to monomeric CTLA-2β. However, thismaterial eluted off of the gel filtration column at approximately 26,000g/mole. This would be the correct molecular weight for a dimer.Therefore, it appears as if CTLA-2β can associate to form non-covalentlylinked dimers and that the dimers can associate to form a tetramer.

The monomeric CTLA-2β from the superdex-75 column was subjected toQ-Sepharose chromatography. The CTLA-2β monomer eluted at 0.32M NaCl andwas separated from the proteolytically cleaved monomer and most of thedimeric species (FIG. 3). Fractions 20-28 were pooled and found to beapproximately 90% pure monomeric CTLA-2β. Fractions 29-34 were used as asource for the dimer. A portion of each of these pools were subjected tochromatography on a reverse-phase C4 column to obtain highly purifiedmaterial for characterization studies (FIG. 4).

Monomer and dimer both eluted at 39% acetonitrile with the onlycontaminants being other CTLA-2β species, as determined by westernanalysis. The monomer sample was approximately 95% pure while the dimersample was contaminated with both monomer and proteolytically modifiedmonomer. All of the characterization studies listed below were carriedout on the reverse phase purified material unless stated otherwise.

                  TABLE 2                                                         ______________________________________                                        AMINO ACID COMPOSITION                                                                                          DIFFER-                                     PMOL       HOI %   NORM    KNOWN  ENCE   % DIFF                               ______________________________________                                        Asp   939.441  13.82   14.91 13     1.91   14.7                               Glu   1067.08  15.70   16.94 16     0.94    5.9                               Ser   503.311  7.41    7.99  8      -0.01  -0.1                               Gly   424.619  6.25    6.74  6      0.74   12.3                               His   130.202  1.92    2.07  2      0.07    3.3                               Arg   458.503  6.75    7.28  7      0.28    4.0                               Thr   380.579  5.60    6.04  6      0.04    0.7                               Ala   496.92   7.31    7.89  8      -0.11  -1.4                               Pro   501.314  7.38    7.96  8      -0.04  -0.5                               Tyr   316.291  4.65    5.02  5      0.02    0.4                               Val   21.089   0.31    0.33         0.33   ERR                                Met   225.013  3.31    3.57  5      -1.43  -28.6                              Cys   0.00     0.00    0.00  0      0.00   ERR                                Ile   81.052   1.19    1.29  1      0.29   28.7                               Leu   500.976  7.37    7.95  8      -0.05  -0.6                               Nlu   980.718                              ERR                                Phe   250.685  3.69    3.98  4      -0.02  -0.5                               Lys   498.543  7.34    7.91  8      -0.09  -1.1                               Trp            0.00    0.00         0.00   ERR                                ______________________________________                                    

Norm MW: 12327.6 Known MW: 12903.0 Accuracy: 95.2% Pmol Analyzed: 63.0NG Analyzed: 813 Pmol Hydrolyzed: 240.8 NG Hydrolyzed: 3107

Amino Acid Analysis of 240.0 pmoles CTLA-2β monomer post C4Reverse-Phase Chromatography.

C) Characterization of CTLA-2β.

(i) Amino Acid Analysis: 240 pmoles of CTLA-2β monomer from C-4chromatography was hydrolyzed and the amino acid composition wasdetermined. The composition had an accuracy of 95.2% when normalized tothe known sequence of CTLA-2β (Table 2, above).

(ii) Circular Dichroism: The CD spectra of monomeric CTLA-2β postQ-Sepharose chromatography (0.226 mg/ml in PBS), monomeric CTLA-2β postC-4 chromatography (0.505 mg/ml in PBS) was taken from 195 to 300 nm,and the resulting spectra are shown in FIG. 5. No difference in thespectra of the monomeric species either before or after reverse-phasechromatography could be found; indicating that treatment withacetonitrile did not effect the secondary structure. However, as listedin Table 3 below, the monomer had 67% B structure while the dimer had53%. The dimer also showed B turn, whereas the monomer did not.Therefore, it appears as if the protein does undergo some structuralchanges upon forming the covalent dimer.

(iii) Mass Spectrophotometry: The mass spectra of monomeric (0.226 mg/mlin PBS) and dimeric CTLA-2β (0.505 mg/ml in PBS) of CTLA-2β from step(iv) of Example 2 was analyzed using a Finigan LASERMAT MassSpectrophotometer. The results are tabulated in Table 3, below. It wasdetermined that the monomeric form of CTLA-2β has a mass of 12.8 kd, andthe dimeric form a mass of 25.4 kd.

                  TABLE 3                                                         ______________________________________                                        CHARACTERIZATION OF CTLA-2β                                                               MONOMER       DIMER                                          ______________________________________                                        Molecular Weight:                                                             on Gel filtration  28     kd       56   kd                                    from non-reducing gel                                                                            14     kd       28   kd                                    from Mass Spec.    12.8   kd       25.4 kd                                    Circular Dichroism:                                                           % Alpha Helix      7.6             8.6                                        % β-turn      0.0             10.9                                       % Random Coil      20.8            27.3                                       % β-sheet     67.4            53.1                                       i.c. 50 in Cathepsin L Assay                                                                     18.2   nM       1.1  nM                                    i.c. 50 in Cathepsin H Assay                                                                     69     nM       NA                                         i.c. 50 in Papain Assay                                                                          14     nM       NA                                         ______________________________________                                    

Summary of the Characterization of monomeric and dimeric CTLA-2β. Themolecular weights of the monomeric and dimeric forms of CTLA-2β weredetermined by the various methods stated. The secondary structure toCTLA-2β was measured using Circular Dichroism and analyzed by comparingthe resulting spectra to standard structures.

(iv) Inhibition of Cathepsin L: The synthetic substrate Z-Phe-Arg-NMec(AMC), was hydrolyzed by Cathepsin L to yield the intensely fluorescent7-amino-4-methylcoumarin. Both monomeric and dimeric forms of CTLA-2β(from reverse-phase chromatography) were shown to have some inhibitoryactivity (FIGS. 6A and 6B).

One characteristic observation is that CTLA-2β did not give 100%inhibition of the proteolytic activity of Cathepsin L even at highconcentrations. The maximum inhibition observed was approximately 60%.This is different from other known inhibitors of Cathepsin L, such asthe cystatins, that will completely inhibit the activity of the enzyme.However, from our data, an i.c. 50 value of 18 nM and 1.1 nM wereobserved for the inhibition of human liver Cathepsin L by monomeric anddimeric CTLA-2β, respectively (Table 3, above). A Ki value of 22 nM wasobtained for the inhibition of human Cathepsin L with CTLA-2β indicatinga tight interaction between these two proteins (FIG. 7).

In order to determine the specificity of CTLA-2β with respect to it'sinhibition of various cysteine proteases, assays were set up for papainas well as Cathepsins H and L (see Example 4, above). The Dixon plot ofthe form V-/V+ vs. I for the inhibition of these proteases by CTLA-2β isshown in FIG. 8. From this plot, the i.c. 50 value has been calculatedto be 14 nM for papain and 69 nM for Cathepsin H. It is interesting tonote that CTLA-2β was able to inhibit 100% of the activity of papaincompared to approximately 60% of the activity of Cathepsin L.

The above data leads to methods of making specific inhibitors of a givencathepsin. Through recombinant DNA techniques, a molecule can beconstructed so that it is highly homologous to the pro-region of theprotease yet retains the functional aspects of the CTLA-2β molecule.

EXAMPLE 5 Synthetic Peptides Derived from CTLA Inhibit Cathepsin LActivity

In an effort to further characterize the inhibitory regions of CTLA, andthe protease inhibiting activity as demonstrated by the previousexamples, four synthetic peptides were generated based on the peptidesequence for CTLA.

p117: YSLDQQRHRRLMWEENKKKIEAH [SEQ ID No. 5]

p118: SLDNEWKEWKTTFAKAYSLDEE [SEQ ID No. 6]

p089: ENKKKIEAHNADYERGKTSFC [SEQ ID No. 7]

p092: CRGEMAPDLPEYEDLG [SEQ ID No. 8]

The peptides were synthesized on a model 430A (Applied Biosystems)peptide synthesizer using NMP-HOBt Fmoc chemistry. The peptides werecleaved and deprotected in 90% TFA, 4% thioanisole, 2% ethanedithiol,and 4% liquefied phenol for 2 hours at room temperature. Afterlyophilization, the peptides were purified by C18 reverse-phasechromatography on a Beckman System Gold HPLC.

These peptides were tested for inhibition of Cathepsin L activity usingthe assays described previously. Briefly, Cathepsin L (19 pM final asdetermined by active site titration using E-64, Barret & Kirschke, 1991)was added to 750 uL of assay buffer consisting of 340 nM sodiumacetate--60 mM acetic acid (pH 5.5), 4 mM disodium EDTA, and 8 mM DTT.Peptides were added at concentrations ranging from on to 35 uM. After a6 minute incubation at 25° C., 1500 uL of 0.1% Brij 35 was added and thereaction started with 5.0 uM Z-Phe-Arg-NMec(AMC) (AMC isaminomethylcoumarin). The fluorescence of the free aminomethylcoumarinwas measured in an SLM 4800s fluorimeter at excitation of 370 nm and anemission at 432 nm over a 6 minute time period. Fluorescence wasconverted to uM AMC released by using a standard curve generated byplotting uM AMC vs. fluorescence, and plotted vs. time. In order tocalculate the velocities, a linear least squares analysis was performedover the initial part of the data. Correlation coefficients were greaterthan 0.98 in all cases. The i.c. 50 values were determined from Dixonplots of the form V-/V+ (velocity without peptide/velocity with peptide)vs. peptide concentration. From these tests, the following results wereobtained.

                  TABLE 4                                                         ______________________________________                                        I.C. 50 DETERMINATION                                                         Peptide          i.c. 50                                                      ______________________________________                                        p117             5.3 uM                                                       p092              41 uM                                                       p089              10 uM inhibited 25%                                         p118             no inhibition at 1000 uM                                     ______________________________________                                    

FIG. 9A is a graph of the data for the assays using the p117 peptide,[SEQ ID No. 5]. FIG. 9B is a Dixon plot derived from this data. Thevarious symbols plot different concentrations of peptide tested (in uM).

FIG. 10A is a graph of the data for the assays using the p092 peptide,[SEQ ID No. 8]. FIG. 10B is a Dixon plot derived from this data. Thevarious symbols plot different concentrations of peptide tested (in uM)as labelled.

FIG. 11A is a graph of the data for the assays using the p089, [SEQ IDNo. 7] peptide. FIG. 11B is a graph of the data for the assays using thep118 peptide, [SEQ ID No. 6]. The various symbols plot differentconcentrations of peptide tested (in uM) as labeled.

From these data it can be seen that the synthetic polypeptide p117 iscapable of effecting the inhibition of cysteine protease activity invitro. The results also indicate varying effectiveness of othersynthetic peptides under similar assay conditions. Thus the p117 peptideis a useful agent for inhibiting cysteine protease activity.

EXAMPLE 6 Synthetic Peptides Derived from CTLA Inhibit ProteoglycanDegradation

Cathepsins B and L have been found to degrade cartilage components,causing the degradation of associated proteoglycans. (Maciewicz andWotton, 1991, Biomed. Biochim. Acta 50:561). Previous results by thesame researchers indicated that these enzymes are found in active formin the synovial fluid of arthritic patients. The conclusion drawn wasthat cysteine proteinases play a role in the etiology of arthritis.

Cartilage degradation can be induced by the in vivo injection of IL-1into the joint capsule of rabbits, and the administration of a largeserine protease inhibitor PN-1 (43 KD) can ameliorate this degradation.It has been found that low molecular weight synthetic peptidemetalloproteinase inhibitor can prevent the breakdown of proteoglycanwithin articular cartilage in vitro. (Andrews et al., 1992, AgentsActions 37:147). Certain cysteine endopeptidase inactivators were foundto inhibit IL-1 stimulated structural cartilage proteoglycandegradation. E64 and Ep475, broad-spectrum cysteine protease inhibitors)did not work at 100 uM concentrations. However lipophillic derivativesinhibited at 10 uM to 1 uM concentrations. (Buttle et al., 1992, ArchBiochem Biophys 299:377). The peptides described in the previous examplewere thus tested for the ability to inhibit articular cartilageproteoglycan degradation, as measured by proteoglycan release after IL-1stimulation.

The assay system used for testing the peptides for inhibitory activityof proteoglycan release is a micro organ culture assay (MOCA). Papain;cetyl pyridinium chloride and chondroitin sulfate type C was purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). Interleukin 1 alpha waspurchased from Collaborative Research. ABCase, chrondroitinase, ABClyase, and keratanase were obtained from ICI. Na ³⁵ SO₄ was purchasedfrom NEN.

Articular cartilage explants from calf knee joints were maintained inculture in DMEM medium containing 20 uCi/ml Na ³⁵ SO₄ for 48 hours forthe incorporation of label into newly synthesized proteoglycan (PG). Theradiolabelled medium was then removed, the radiolabelled explants washed3×30 ml cold DMEM and placed into a 96 well plate with or without IL-1(Interleukin-1 alpha, 50 U/ml) and various concentrations of p117. Theexplants were incubated first for 24 hours in the presence of IL-1 inorder to ensure initiation of IL-1 induced auto-catalysis prior to theaddition of various metalloprotease inhibitors for an additional 72hours.

The newly synthesized radiolabelled proteoglycans released during thecultivation period were subjected to enzymatic digestion with papain.Briefly, an aliquot of 150 ul of medium from the original culture volume(300 ul) was incubated with 100 ul of papain (3 mg/ml) for 2 hours at65° C. A 50 ul aliquot of the papain digested material containingradiolabelled ³⁵ SO₄ -gag (glycosaminoglycans, mucopolysaccharide) wasincubated with 100 ul of cetylpyridinium chloride (cpc, 4% cpc+40 nMNaSO₄) plus cold chondroitin sulfate standard (30 ul of 2.5 mg/mlsolution). The samples were placed on ice for 60 minutes, and theradiolabelled gags were precipitated and collected on a 96 well plateharvester (MACH2, TOMTEC, Orange, CT) glass fiber filter. The filter wasdried and counted after addition of 10 mls of scintillation cocktail(scintillant).

FIG. 12 is a graph showing inhibition of proteoglycan release from MOCAby p117 peptide. The ability of p117 to inhibit the IL-1 induceddegradation of articular cartilage was shown to be best at aconcentration of p117 of 10 uM. The resulting inhibition is about 30%.

These results indicate that the synthetic peptide p117, acting on theactivity of Cathepsin L and/or Cathepsin B is effective as an inhibitorof proteoglycan release in situ. The MOPC assay is an acceptable modelsystem for in vivo disease of the connective tissues, and indicate thatthe p117 polypeptide would be an effective agent for use in vivo.

EXAMPLE 7 Theoretical Minimum Inhibitory Polypeptide Conserved Sequence

Examination of the human Cathepsin L proregion sequence (SEQ ID NO. 12)and the mouse Cathepsin L proregion sequence (SEQ ID NO. 13) revealsregions of homolgy. This combined with the findings of the activity ofthe peptides of the present invention lead to the conclusion that therecould be a minimum conserved sequence that will maintain inhibitoryactivity.

Taking into account that the best activity was found for p117, (residues26-47), the partial activity of p089 (residues 39-58), and the pooractivity of p118 (residues 9-30), the comparison narrowed down the idealconserved sequence as set out in the table below.

Human CATL TLTFDHSLEAQWTKW KAMHNRLYGMNEEGW RRAVWEKNMKMIELH N

Mouse CATL DQTF--S--AEWHQW KSTHRRLYGTNEEEW RRAIWEKNMRMIQLH N

CTLA B ----DPSLDNEWKEW KTTFAKAYSLDEERH RRLMWEENKKKIEAH N

huCATL QEYREGKHSFTMAM NAFGDMTSEEFRQVM NGFQNRKPRKGKVFQ EPLFYE

muCATL GEYSNGQHGFSMEM NAFGDMTNEEFRQVV NGYRHQKHKKGRLFQ EPLMLK

CTLA B ADYERGKTSFYMGL NQFSDLTPEEFRTNC CGSSMCRGEMAPDLP EYEDLG

CTLA B KNSYLTPGRAQPE

The high-lighted segment corresponds to what is believed to be theminimal conserved inhibition sequence. We would predict that thesepeptide fragments will act as effective inhibitory peptides.

Human CATL RRAVWEKNMKMIELHN (SEQ ID NO. 9)

Mouse CATL RRAIWEKNMRMIQLHN (SEQ ID NO. 10)

CTLA B RRLMWEENKKKIEAHN (SEQ ID NO. 11)

The advantage of using the peptide sequence that is based upon the humansequence is that it may be more specific, and perhaps lack antigenicityin human patients.

The invention and the manner and process of making and using it, are nowdescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the spirit or scope of the presentinvention as set forth in the claims. To particularly point out anddistinctly claim the subject matter regarded as the invention, thefollowing claims conclude this specification.

    __________________________________________________________________________    #              SEQUENCE LIS - #TING                                           - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 13                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 141 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..141                                                #/note= "clta-2b from Denizot et                                                             al., Eur. - # J. Immunol. 19:631-635 (1989)"                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Leu Asp Asn Lys Val Leu Val Ser Ile Cys Gl - #u Gln Lys Leu Gln His         #                15                                                           - Phe Ser Ala Val Phe Leu Leu Ile Leu Cys Le - #u Gly Met Met Ser Ala         #            30                                                               - Ala Pro Ser Pro Asp Pro Ser Leu Asp Asn Gl - #u Trp Lys Glu Trp Lys         #        45                                                                   - Thr Thr Phe Ala Lys Ala Tyr Ser Leu Asp Gl - #u Glu Arg His Arg Arg         #    60                                                                       - Leu Met Trp Glu Glu Asn Lys Lys Lys Ile Gl - #u Ala His Asn Ala Asp         #80                                                                           - Tyr Glu Arg Gly Lys Thr Ser Phe Tyr Met Gl - #y Glu Asn Gln Phe Ser         #                95                                                           - Asp Leu Thr Pro Glu Glu Phe Arg Thr Asn Cy - #s Cys Gly Ser Ser Met         #           110                                                               - Cys Arg Gly Glu Met Ala Pro Asp Leu Pro Gl - #u Tyr Glu Asp Leu Gly         #       125                                                                   - Lys Asn Ser Tyr Leu Thr Pro Gly Arg Ala Gl - #n Pro Glu                     #   140                                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "5' primer for ctla-2b" /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #                24CTCC ATCC                                                  - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "3' primer for clta-2b" /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #  42              TCGG TCTCATTCGA TCGCCTAGGG GG                              - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 111 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Ala Ala Pro Ser Pro Asp Pro Ser Leu As - #p Asn Glu Trp Lys Glu         #                15                                                           - Trp Lys Thr Thr Phe Ala Lys Ala Tyr Ser Le - #u Asp Glu Glu Arg His         #            30                                                               - Arg Arg Leu Met Trp Glu Glu Asn Lys Lys Ly - #s Ile Glu Ala His Asn         #        45                                                                   - Ala Asp Tyr Glu Arg Gly Lys Thr Ser Phe Ty - #r Met Gly Leu Asn Gln         #    60                                                                       - Phe Ser Asp Leu Thr Pro Glu Glu Phe Arg Th - #r Asn Cys Cys Gly Ser         #80                                                                           - Ser Met Cys Arg Gly Glu Met Ala Pro Asp Le - #u Pro Glu Tyr Glu Asp         #                95                                                           - Leu Gly Lys Asn Ser Tyr Leu Thr Pro Gly Ar - #g Ala Gln Pro Glu             #           110                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..23                                                 #/note= "p117 SYNTHETIC PEPTIDE"                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Tyr Ser Leu Asp Glu Glu Arg His Arg Arg Le - #u Met Trp Glu Glu Asn         #                15                                                           - Lys Lys Lys Ile Glu Ala His                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..22                                                 #/note= "p118 SYNTHETIC PEPTIDE"                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Ser Leu Asp Asn Glu Trp Lys Glu Trp Lys Th - #r Thr Phe Ala Lys Ala         #                15                                                           - Tyr Ser Leu Asp Glu Glu                                                                 20                                                                - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..21                                                 #/note= "p089 SYNTHETIC PEPTIDE"                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Glu Asn Lys Lys Lys Ile Glu Ala His Asn Al - #a Asp Tyr Glu Arg Gly         #                15                                                           - Lys Thr Ser Phe Cys                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..16                                                 #/note= "p092 SYNTHETIC PEPTIDE"                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Cys Arg Gly Glu Met Ala Pro Asp Leu Pro Gl - #u Tyr Glu Asp Leu Gly         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Arg Arg Ala Val Trp Glu Lys Asn Met Lys Me - #t Ile Glu Leu His Asn         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Arg Arg Ala Ile Trp Glu Lys Asn Met Arg Me - #t Ile Gln Leu His Asn         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Arg Arg Leu Met Trp Glu Glu Asn Lys Lys Ly - #s Ile Glu Ala His Asn         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 96 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..96                                                 #/note= "huCATL"HER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Thr Leu Thr Phe Asp His Ser Leu Glu Ala Gl - #n Trp Thr Lys Trp Lys         #                15                                                           - Ala Met His Asn Arg Leu Tyr Gly Met Asn Gl - #u Glu Gly Trp Arg Arg         #            30                                                               - Ala Val Trp Gly Lys Asn Met Lys Met Ile Gl - #u Leu His Asn Gln Glu         #        45                                                                   - Tyr Arg Glu Gly Lys His Ser Phe Thr Met Al - #a Met Asn Ala Phe Gly         #    60                                                                       - Asp Met Thr Ser Glu Glu Phe Arg Gln Val Me - #t Asn Gly Phe Gln Asn         #80                                                                           - Arg Lys Pro Arg Lys Gly Lys Val Phe Gln Gl - #n Pro Leu Phe Tyr Glu         #                95                                                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 92 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..92                                                 #/note= "muCATL"HER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Asp Gln Thr Phe Ser Ala Glu Trp His Gln Tr - #p Lys Ser Thr His Arg         #                15                                                           - Arg Leu Tyr Gly Thr Asn Glu Glu Glu Trp Ar - #g Arg Ala Ile Trp Glu         #            30                                                               - Lys Asn Met Arg Met Ile Gln Leu His Asn Gl - #y Glu Tyr Ser Asn Gly         #        45                                                                   - Gln His Gly Phe Ser Met Glu Met Asn Ala Ph - #e Gly Asp Met Thr Asn         #    60                                                                       - Glu Glu Phe Arg Gln Val Val Asn Gly Tyr Ar - #g His Gln Lys His Lys         #80                                                                           - Lys Gly Arg Leu Phe Gln Gln Pro Leu Met Le - #u Lys                         #                90                                                           __________________________________________________________________________

What we claim:
 1. A polypeptide having an amino acid sequence selectedfrom the group consisting of the amino acid sequence of SEQ ID NO:5(p117), SEQ ID NO:6 (p118), SEQ ID NO:7 (p089), and SEQ ID NO:8 (p092).2. A polypeptide having an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO:9 (huCATL homolog),SEQ ID NO:10 (muCATL homolog), and SEQ ID NO:11 (CTLA B homolog).
 3. Apolypeptide dimer, wherein at least one polypeptide dimer component is apolypeptide having the amino acid sequence of SEQ ID NO:4, and thesecond polypeptide dimer component is a polypeptide of claim
 1. 4. Apolypeptide dimer of claim 3, wherein said dimer has an apparentmolecular weight of between 22 and 28 kDa as measured by SDS PAGE.
 5. Amethod for inhibiting the proteolytic activity of a cysteine protease invitro comprising contacting said cysteine protease with an effectiveinhibiting amount of at least one polypeptide which has an amino acidsequence selected from the group consisting of the amino acid sequenceof SEQ ID NO:5 (p117), SEQ ID NO:7 (p089), SEQ ID NO:8 (p092), SEQ IDNO:11 (CTLA B homolog), SEQ ID NO:9 (huCATL homolog), and SEQ ID NO:10(muCATL homolog).
 6. The method of claim 5, wherein the cysteineprotease is selected from the group consisting of Cathepsin H, CathepsinL, and papain.
 7. A method for inhibiting the degradation ofproteoglycans in vitro comprising administering an effective inhibitingamount of at least one polypeptide which has the amino acid sequenceselected from the group consisting of the amino acid sequence of SEQ IDNO:5 (p117), SEQ ID NO:7 (p089), SEQ ID NO:8 (p092), SEQ ID NO:11 (CTLAB homolog), SEQ ID NO:9 (huCATL homolog), and SEQ ID NO:10 (muCATLhomolog).